Immunoassay techniques have been known for the last few decades and are now commonly used in medicine for a wide variety of diagnostic purposes to detect target analytes in a test sample. Immunoassays exploit the highly specific binding of an antibody to its corresponding antigen, wherein the antigen is the target analyte. Typically, quantification of either the antibody or antigen is achieved through some form of labeling such as radio- or fluorescence-labeling. Sandwich immunoassays involve binding the target analyte in the sample to the antibody site (which is frequently bound to a solid support), binding labeled antibody to the captured analyte, and then measuring the amount of bound labeled antibody, wherein the label generates a signal proportional to the concentration of the target analyte inasmuch as labeled antibody does not bind unless the analyte is present in the sample.
Immunoassay methods can be carried out in any of a wide variety of formats. A typical heterogeneous sandwich immunoassay employs a solid phase as a support to which is bound a first (capture) antibody reactive with at least one epitope on the target analyte. A second (detection) antibody is also reactive with at least one epitope the target analyte, and may be conjugated to a detectable label that provides a signal that is measured after the detection antibody binds to the captured target analyte.
PIVKA-II (“Protein Induced by Vitamin K Absence or Antagonist-II”), also known as des-carboxy prothrombin (DCP), is a tumor marker useful for diagnosing and determining the prognosis of hepato-cellular carcinoma (HCC). PIVKA-II is also present in vitamin K deficiency or in patients using warfarin. Sensitivity of PIVKA-II detection methods can be important for establishing a diagnosis before clinical symptoms occur. Immunoassays for PIVKA-II are available, yet nonspecific binding interactions between the antibodies used and antigens other than PIVKA-II that may be present in a sample, reduce sensitivity and accuracy around the cutoff levels typically employed. Nonspecific antibody/antigen interactions can increase the risk of false positive diagnostic results and the risk that individuals will not obtain a diagnosis that is both timely and accurate. Methods for improving the sensitivity and accuracy of PIVKA-II immunoassays are therefore needed.